In the United States alone, annual influenza epidemics cause approximately 30,000 deaths each year, mostly among the elderly. In addition, periodic pandemics result from the emergence of novel influenza viruses through antigenic shift. In pandemic years, the virus efficiently infects most of the human population due to a lack of pre-existing immunity against the hemagglutinin of the pandemic strain. Three influenza pandemics have occurred in the last century, in 1918, 1957 and 1968, and in each case mortality and morbidity far exceeded those in epidemic years. In fact, the most severe of the three pandemic episodes (in1918) is estimated to have caused greater than 40 million deaths. Although it is not possible to predict when the next influenza pandemic will occur, that there will be a new pandemic is almost certain. The potentially devastating effects of this event could be prevented with the fast production and distribution of an effective vaccine designed to protect against the pandemic virus. Prototype pandemic vaccines developed to date require multiple high doses to induce protective responses in humans. With such a vaccine, it would be impossible to generate sufficient supplies to promptly immunize the entire population using current manufacturing technology. In addition, more effective vaccines are needed to control epidemic influenza in high risk populations, where the success rate of vaccination with currently approved vaccines is low. We propose to investigate a novel concept for the design of improved influenza virus vaccines based on modification of the viral NS1 gene to generate live attenuated vaccine strains. We have previously shown that the NS1 gene of influenza virus is responsible for inhibition of type I IFN production. We therefore hypothesize that NS1-modified influenza virus vaccines will be attenuated and will stimulate potent innate and adaptive immune responses due to their IFN-inducing properties. Thus, H3N2 and H5N1 viruses containing truncations of their NS1 genes will be generated by reverse genetics as prototype vaccines against epidemic and pandemic influenza in humans. The recombinant viruses will be evaluated in preclinical models for their ability to protect against influenza virus infection. In addition, H5N1-based vaccines will be tested for their ability to protect poultry, in order to assess their utility on poultry farms. The immune responses induced by NS1-modified influenza virus vaccines will be characterized in detail, providing insight into the mechanisms of virulence of wild-type influenza virus strains. We hypothesize that the NS1 protein of influenza virus is responsible for a suboptimal immune response after virus infection, and that the NS1- muntant viruses will have increased immunogenicity. We will also explore innovative approaches to eliminate the potential for reassortment between live vaccine viruses and circulating strains that could result in the acquisition of virulence. Finally, in collaboration with GreenHills Biotechnology, we will optimize cell culture techniques for the large-scale production of NS1-modified influenza virus vaccines suitable for use in humans.